All multicellular organisms originate from a small set of pluripotent embryonic stem cells that expand and differentiate into tissues and organs of a mature individual, and organisms from worms to humans use a highly conserved set of core developmental pathways to complete the process of embryogenesis. However, in adults during normal growth and aging, or after injury, differentiated cells and organs must be replenished or regenerated. Regeneration is carried out by long-lived, usually lineage-restricted stem cells that retain the capacity to expand and differentiate throughout the lifespan of the individual. However, the cellular and molecular mechanisms underlying regenerative development of most tissues are not well understood. More importantly, despite the deep conservation of embryonic developmental pathways, the degree to which different organisms can regenerate tissues and organs following injury is not a conserved feature throughout evolution: a salamander can regenerate an amputated limb, but a human cannot. Why is this true? This grant is focused on studying the regenerative abilities of a basal chordate organism, the colonial ascidian Botryllus schlosseri, to explore these questions. Using a simple surgical procedure, we can induce Botryllus to regenerate an entire body, including a heart, GI tract, pharynx, vasculature, neural and endocrine system- and gametes, from fragments of an extracorporeal vascular bed. This process, called whole body regeneration (WBR), occurs rapidly (3-6 days), and ascidians are the only chordates that can regenerate entire bodies, thus we can dissect how highly conserved developmental pathways are redeployed during regeneration of any tissue. Botryllus also provides unique ways to study WBR, for example, the vasculature is sessile and transparent, and easy to visualize, label and manipulate. Importantly, we have also recently developed a rescue/reconstitution assay that will allow us to prospectively isolate the cells responsible, as well as characterize and functionally assess developmental pathways underlying WBR. This R21 grant is designed to develop this new system, and we propose to: 1) utilize limiting dilution transplantation assays and cell labeling transplantation strategies to assess whether progenitor cells are lineage restricted or pluripotent; and, 2) use gene expression profiling of proliferating cells at different stages during regeneration to identify genes and signaling pathways that regulate stem cell function and induce recruitment and proliferation of circulatory stem cells. We will then functionally test the role of a subset of regenerative pathways and gene regulatory mechanisms using small molecule inhibitors and RNAi. Our goals are that by the end of this 2-year grant, we will have completed a large database of differentially expressed genes, completed initial genetic and functional characterization the developmental pathways underlying regeneration, as well as either having isolated, or have a robust methodology to purify the cells responsible and dissect their role in regeneration. By characterizing the stem cell population responsible for whole body regeneration and identifying genes, signaling pathways and the microenvironment that regulate stem cell recruitment, proliferation and differentiation during regenerative growth, these experiments will greatly advance Botryllus as a model system for the study of genes conserved in stem cell and regenerative biology, and will advance our understanding of human stem-cell function and tissue regeneration.